The overall goal of this project is to determine the mechanisms by which radiation therapy (RT) of tumors stimulates anti-tumor immunity that can ultimately result in tumor control/cure. Our studies during the last grant period illustrated the pivotal role of IFN? responsiveness to RT in several tumor model systems. One particularly interesting finding in the Colon 38 mouse tumor model was that a single high dose (15 Gy) of local RT results in tumor control in approximately half of the tumors (responders), whereas in the other mice the tumor continues to grow progressively (non-responders). This mimics the clinical situation in which RT, although often extremely effective, nevertheless does not work in all patients. We demonstrated that the immune system and two factors in particular, IFN? and CD8+ T cells, are crucial for the responder phenotype. Having demonstrated the effectiveness of a single high dose of irradiation, we will now determine how radiation protocols more similar to those used clinically including hyper- and hypo-fractionation schemes affect the levels of IFN? and the number and type of immune cells within the tumor microenvironment (Aim 1). Physical factors within the tumor microenvironment, particularly low oxygenation (hypoxia) are known to contribute to radiation resistance. Additionally, our preliminary data have identified a novel immunosuppressive role for hypoxia in inhibiting IFN? responsiveness, an important property that may also dictate RT efficacy. Therefore, we will use the EF5 hypoxia detection system and an innovative imaging strategy as well as flow cytometry to directly examine the extent of hypoxia within untreated and RT treated tumors. Furthermore, we will determine if these hypoxic niches and the immune cell types found within are non-responsive to IFN? and therefore escape destruction by both RT and the immune system. Thus, we will examine the location and functional ability of T cells in both the hypoxic and normoxic regions. We will determine how the different modes of RT delivery alter the extent of hypoxia within treated tumors and whether or not this results in greater effectiveness of the immune system (Aim 2). Because hypoxia within tumors seems to protect tumor cells from radiation therapy and attack by immune effector cells, we will determine if the cytokine, IL-12 can alleviate the extent of hypoxia and improve immune function (Aim 3). This cytokine is particularly powerful because it can attenuate some of the abnormalities in tumor blood vessels making them more functional. It addition, it is a strong inducer of cellular immunity, which results in enhanced production of IFN? and a more effective anti-tumor response. These studies will add essential mechanistic understanding to how RT and the immune system interact and allow optimal translation to clinical treatment of tumors.